One major family of endogenous antibacterial peptides, the beta-defensins, are secreted by the epithelial cells that line the digestive, respiratory and urogenital tracts of higher mammals. They are also produced by keratinocytes within the skin. Their primary role is to provide an essential first line of defence against infection via these routes by pathogenic organisms.
The defensins are one of the most studied classes of antimicrobial peptides. This class consists of cysteine-rich molecules with three disulphide bridges. They are found in plants, insects and various mammals. In humans, two classes of defensin are found which differ from one another in terms of spacing and bonds between the six cysteine residues. The first of these classes is the alpha-defensins (six types) that have been isolated from neutrophils (HNP1-4, human neutrophil peptide) and in the paneth cells of the gastrointestinal tract (alpha-defensins 5 and 6). The second class, the beta-defensins, are longer, more basic, and are expressed throughout the mucosae within the epithelial cells and keratinocytes that line and/or comprise and/or are present within the digestive, respiratory and urogenital tracts and the skin. hBD1 (human beta-defensin 1) is secreted constitutively and human beta-defensins 2, 3 and 4 (hBD2, hBD3 and hBD4) are produced in response to infection or inflammation. hBD2 expression and secretion is triggered by bacterial stimulation, particularly flagellated bacteria (Harder et al, Nature 1997; 387:861) and IL1 [alpha] and IL1 [beta] (Interleukin 1) (Liu et al, J Invest Dermatol 2002; 118; 275-281). In some tissue sites, Tumour Necrosis factor alpha (TNF-alpha) and Lipopolysaccharide (LPS) may also play a role in inducing hBD2 expression. In vitro experiments have revealed that hBD2 is active against Gram negative bacteria such as Escherichia coli (E. coli) and to a lesser extent, Gram positive bacteria such as Streptococcus pneumoniae (Str. pneumoniae). hBD2 also demonstrates killing activity in vitro against the yeast Candida albicans. hBD3 expression and secretion is induced by bacterial stimulation, TNF-alpha and especially Interferon gamma (IFNγ) which also have the common property of being molecules involved in inflammatory processes.
In addition to the potent, constitutive and regulated broad-spectrum innate antimicrobial protection the beta-defensins provide, these molecules, hBD2 in particular, also have the ability to mobilise the adaptive arm of the immune response through chemotactic effects on immature dendritic cells and memory T-lymphocytes (Yang et al, Science 1999; 286: 525-528).
Importantly, evidence is coming to light that beta-defensins not only provide defence against infection from pathogenic microbes, but are key in regulating and maintaining optimal density and diversity of the body's essential commensal microbial ecosystems, such as those on the skin, and within the gastrointestinal and genital tracts (Ganz, T, Nat Rev Immunol. 2003 3(9): 710-20).
The mode of action of beta-defensins is such that they are largely non-toxic to host cells at active concentrations. The beta-defensins have, therefore, been implicated as potential targets for therapeutics for a wide range of infections. However, natural forms of defensins are technically challenging to produce in recombinant systems resulting in low yields. Moreover, evidence is growing to suggest that through their chemotactic actions, beta-defensins are potent inflammatory compounds (Yang et al, Science 1999; 286: 525-528; Van Wetering et al. Inflamm Res. 2002; 51(1): 8-15; Niyonsaba et al. Curr Drug Targets Inflamm Allergy 2003; 2(3): 224-231). Taken together, these factors make natural defensins unsuitable for therapeutic applications.
Beta-defensins are also highly salt sensitive (Porter et al., Infect. Immun. 1997; 65(6): 2396-401; Bals et al. J Clin Invest. 1998; 102(5): 874-80; Valore et al., J Clin Invest 1998; 101(8): 1633-42; Goldmann et al., Cell 1997; 88(4): 553-609; Singh et al., Proc Natl Acad Sci USA 95(25): 14961-6). For this reason, beta-defensins cannot provide antimicrobial protection in conditions such as cystic fibrosis wherein, although the respiratory epithelia produce abundant beta-defensins in response to the persistent bacterial infections associated with this condition, they are inactive due to the imbalance in ion transport across the respiratory epithelial membranes that results in increased cation resorption (Na+ in particular) and increased chloride secretion (Donaldson S H and Boucher R C. Curr. Opin. Pulm. Med. 2003 November; 9(6):486-91; Davies J C. Pediatr Pulmonol Suppl. 2004; 26:147-8.)
There is a requirement, therefore, for further agents that can be used to treat microbial infections.
The present inventors have identified peptides that, surprisingly, have improved antimicrobial activity over natural defensins.